mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Visual evidence for horizontal gene transfer between plants
One of the major concerns with cultivating genetically modified plants is the potential risk of horizontally transfering resistance genes into other organisms within the environment. The fate of transgenic DNA released during plant decomposition is an area of heated debate, as recent studies have shown that a sizable fraction can be stably detected in the soil several years after a gentically modified plant is cultivated 1. Under these conditions, it is thought that the greatest risk of gene transfer occurs within the microbial communities actively degrading the plant matter. Of the three modes of horizontal gene transfer found in prokaryotes, natural transformation or the ability of a cell to take up foreign DNA from the environment, is thought to be the only feasible means of transferring DNA from plants to bacteria 3. In a recent study by Pontiroli'' et al''', '''the authors looked for direct visual evidence of horizontal gene transfer between ''Acinetobacter baylyi and transplastomic tobacco plants using an A. baylyi ''strain carrying a promoterless GFP reporter plasmid and spectinomycin-restant plants. Additionally, the authors examined whether certain regions of the plant commonly colonized by the bacterium were more susceptible for permitting horizontal gene transfer. The methods and major findings from this study are described in greater detail below. ' ' Methods Development of a transformation reporter In order to visualize the transfer of transgenic plant DNA into ''Acinetobacter baylyi, ''the authors engineered a reporter system that allows for the detection of homologous recombination driven gene transfer events between the bacteria and DNA engineered into the tobacco chloroplast. In this system, a strain of ''A. baylyi ''is utilized that carries a promoterless copy of ''gfp incorporated into the chromosome flanked by two cholorplast genes, rbcL & the 3' end of accD. In conjunction, the spectinomycin-resistance gene under the control of a bacterial promoter was engineered inbetween these two genes on the chloropast chromosome of the transplastomic tobacco plant. As a result, if homologous recombination occurs between the bacterial chromosome and the chloroplast DNA at these shared sequences, fluorescent, spectinomycin resistant transformants will be produced (Schematic shown at right). Results Growth of ''Acinetobacter ''on plant tissues The authors first determined whether the bacteria in question preferentially colonized different regions of the plant. Through several growth assays, they were able to determine that ''Acinetobacter ''exhibited the poorest growth rates on whole tobacco leaves, and grew optimally around the roots and vascular tissues of the plant. However, exponential growth was only observed when the leaves were crushed to mimick decaying plant matter. Competence development ''in vitro ''and on ground plant tissues The authors next assessed the competence of the engineered ''A. baylyi ''strain under two conditions. In one scenario, these cells were grown on LB agar in the presence of purifed transgenic tobacco DNA, and in the other condition, the bacteria was grown on crushed tobacco leaves supplemented with tobacco leaf DNA. Spectinomycin resistant, flourescent transformants were observed in both growth conditions. Interestingly, it was found that transformation rates were highest during exponential growth. Natural transformation of ''Acinetobacter baylyi '' The authors next looked at whether the bacteria could become naturally transformed solely through growth on whole plant tissues. After seven days, fluorescent cells were clearly observed on all tissues tested, suggesting that horizontal gene transfer from the transgenic plants was readily occuring. Summary of results As a whole, the results from this study suggest that ''Acinetobacter baylyi ''is naturally competent and capable of taking up transgenic DNA from tobacco plants. Transformed ''Acinetobacter baylyi ''cells were visually detected on the plant through fluorescence microscopy, and successfully selected for by culturing on media containing spectinomycin. The authors were also able to determine that cell competence increased as the bacterial population approached the exponential growth phase, and that competence also increased when ''A. baylyi ''was cultured in the presence of pathogenic bacteria-likely as a result of plant cell lysis. Additionally, the researchers were able to determine that several "hotspots" for transformation existed within the plant's biomass, and were typically found at the root surface or sites of physical trauma. References 1 Ceccherini, M. T., J. Pote, E. Kay, V. T. Van, J. Marechal, G. Pietramellara, P. Nannipieri, T. M. Vogel, and P. Simonet. 2003. Degradation and transformability of DNA from transgenic leaves. Appl. Environ. Microbiol. 69:673-678. 2 Bertolla, F., and P. Simonet. 1999. Horizontal gene transfers in the environment: natural transformation as a putative process for gene transfers between transgenic plants and microorganisms. Res. Microbiol. 150:375-384. 3 Nielsen, K. M., A. M. Bones, K. Smalla, and J. D. van Elsas. 1998. Horizontal gene transfer from transgenic plants to terrestrial bacteria—a rare event? FEMS Microbiol. Rev. 22:79-103. 4 Alessandra Pontiroli et al. Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco. Appl Environ Microbiol. 2009 May; 75(10): 3314–3322. 5 http://en.wikipedia.org/wiki/Acinetobacter